Similar articles

Abstract: Good quality deoxyribonucleic acid (DNA) is the pre-requisite for its downstream applications. The presence of high concentrations of polysaccharides, polyphenols, proteins, and other secondary metabolites in mango leaves poses problem in getting good quality DNA fit for polymerase chain reaction (PCR) applications. The problem is exacerbated when DNA is extracted from mature mango leaves. A reliable and modified protocol based on the cetyltrimethylammonium bromide (CTAB) method for DNA extraction from mature mango leaves is described here. High concentrations of inert salt were used to remove polysaccharides; Polyvinylpyrrolidone (PVP) and β-mercaptoethanol were employed to manage phenolic compounds. Extended chloroform-isoamyl alcohol treatment followed by RNase treatment yielded 950‒1050 µg of good quality DNA, free of protein and RNA. The problems of DNA degradation, contamination, and low yield due to irreversible binding of phenolic compounds and coprecipitation of polysaccharides with DNA were avoided by this method. The DNA isolated by the modified method showed good PCR amplification using simple sequence repeat (SSR) primers. This modified protocol can also be used to extract DNA from other woody plants having similar problems.

[1]Bompard, J.M., 1993. The genus Mangifera rediscovered: the potential contribution of wild species to mango cultivation. Acta Hort., 341:69-77.

[2]Dabo, S.M., Mitchell, E.D., Melcher, U., 1993. A method for the isolation of nuclear DNA from cotton (Gossypium) leaves. Anal. Biochem., 210(1):34-38.

[3]Davis, T.M., Yu, H., Haigis, K.M., McGowan, P.J., 1995. Template mixing: a method of enhancing detection and interpretation of codominant RAPD markers. Theor. Appl. Genet., 91(4):582-588.

[4]Dellaporta, S.L., Wood, J., Hicks, J.B., 1983. A plant DNA mini-preparation: version 2. Plant Mol. Biol. Rep., 1(4):19-21.

[5]Doyle, J.J., Doyle, J.L., 1987. A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochem. Bull., 19:11-15.

[6]Doyle, J.J., Doyle, J.L., 1990. Isolation of plant DNA from fresh tissue. Focus, 12:13-15.

[7]Duval, M.F., Bunel, J., Sitbon, C., Risterucci, A.M., 2005. Development of microsatellite markers for mango (Mangifera indica L.). Mol. Ecol. Notes, 5(4):824-826.

[8]Fang, G., Hamrnar, S., Grumet, R., 1992. A quick and inexpensive method for removing polysaccharides from plant genomic DNA. Biofeedback, 13(1):52-54.

[9]John, M.E., 1992. An efficient method for isolation of RNA and DNA from plants containing polyphenolics. Nucleic. Acid Res., 20(9):2381.

[10]Kotchoni, S.O., Gachomo, E.W., Betiku, E., Shonukan, O.O., 2003. A home made kit for plasmid DNA mini preparation. Afr. J. Biotechnol., 2(4):88-90.

[11]Lodhi, M.A., Daly, M.A., Weeden, N.F., Reisch, B.I., 1995. A molecular marker based linkage map of Vitis. Genome, 38(4):786-794.

[12]Manoj, K., Tushar, B., Sushama, C., 2007. Isolation and purification of genomic DNA from Black Plum (Eugenia jambolana Lam.) for analytical applications. Int. J. Biotechnol. Biochem., 3(1):49-55.

[13]Moller, E.M., Bahnweg, G., Sandermann, H., Geiger, H.H., 1992. A simple and efficient protocol for isolation of high molecular weight DNA from filamentous fungi, fruit bodies and infected plant tissues. Nucleic Acids Res., 20(22):6115-6116.

[14]Padmalatha, K., Prasad, M.N.V., 2006. Optimization of DNA isolation and PCR protocol for RAPD analysis of selected medicinal and aromatic plants of conservation concern from Peninsular India. Afr. J. Biotechnol., 5(3):230-234.

[15]Pirttilä, M.A., Hirsikorpi, M., Kämäräinen, T., Jaakola, L., Hohtola, A., 2001. DNA isolation methods for medicinal and aromatic plants. Plant Mol. Biol. Rep., 19(3):273.

[16]Porebski, S., Bailey, L.G., Baum, B.R., 1997. Modification of a CTAB DNA extraction protocol for plants containing high polysaccharide and polyphenol components. Plant Mol. Biol. Rep., 15(1):8-15.

[17]Saghai-Maroof, M.A., Soliman, K.M., Jorgensen, R.A., Allard, R.W., 1984. Ribosomal DNA spacer-length polymorphism in barley: Mendelian inheritance, chromosomal location, and population dynamics. PNAS, 81(24):8014-8019.

[18]Sambrook, J., Fritsch, E.F., Maniatis, T., 1989. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring, Harbour, New York, USA.

[19]Schnell, R.J., Knight, R.J.Jr., 1992. Frequency of zygotic seedlings from polyembryonic mango rootstocks. HortScience, 27(2):174-176.

[20]Schnell, R.J., Olano, C.T., Quintanilla, W.E., Meerow, A.W., 2005. Isolation and characterization of 15 microsatellite loci from mango (Mangifera indica L.) and cross-species amplification in closely related taxa. Mol. Ecol. Notes, 5(3):625-627.

[21]Sharma, A.D., Gill, P.K., Singh, P., 2002. DNA isolation from dry and fresh samples of polysaccharide-rich plants. Plant Mol. Biol. Rep., 20(4):415.

[22]Singh, R.N., 1996. Mango. ICAR, New Delhi.

[23]Zhang, J., McD. Steward, J.D., 2000. Economical and rapid method for extracting cotton genomic DNA. J. Cotton Sci., 4(3):193-201.